This invention relates generally to improvements in apparatus and methods for simultaneously analyzing a plurality of fluid samples to determine the concentration of one or more solid components contained within each sample. More specifically, the present invention relates to an assay cartridge and method for conducting an immunoassay using the assay cartridge to dilute a plurality of blood samples simultaneously and to present the diluted samples for analysis by an imaging instrument.
The recent proliferation of diagnostic tests for an increasing variety of clinically significant target components has created a demand for routine monitoring of these components in patient samples. For example, the blood concentration of T-lymphocytes expressing the CD4 or CD8 surface antigens is widely accepted as a reliable indicator of disease stage in individuals diagnosed with the human immunodeficiency virus (HIV). The need for a cost effective reliable method for routine analysis has led to the development of single-use assay cartridges.
In such a cartridge, a small volume of blood sample is applied to the cartridge by an operator of an analytical instrument. The cartridge is then inserted in the instrument which automatically performs the remaining assay steps. The quantity of reagents used is minimized and the potential for operator error or exposure to biohazardous materials is greatly reduced when such assay cartridges are used. Such cartridges are readily adapted for various assay methods, such as transporting and metering of the sample or reagent, dilution of the sample and presentation of the sample for analysis.
Numerous assays have been developed for identifying a variety of target components found in biological samples. In such assays, a biological sample, e.g. blood or urine, is reacted with a reagent which modifies the component to be detected. Examples of reagents commonly used include binding agents and ligands such as monoclonal antibodies, degradative agents such as protease, and labels such as fluorescent dyes, u.v. active and radioactive compounds. Frequently, the reagent is a monoclonal antibody bound to a fluorescent dye. An imaging instrument is used for quantitative and qualitative analysis of a mixture of the sample and reagent. Once the sample is mixed and incubated with the reagent, an aliquot of the mixture is then isolated and analyzed for the presence or absence of the target component. Immunoassays on blood samples where fluorescent tagged antibodies are used to bind to specific blood cells are examples of such assays.
For an aliquot of an assay sample to be representative of a biological sample as a whole, it is important that the target component be evenly distributed within the sample when analyzed. The assay process should not create an uneven distribution of the component within the sample. Some prior art cartridges and assays, however, subject the sample to large centrifugal forces which disrupt the distribution of the components within the sample. Similarly, larger target components in suspension in the sample, such as blood cells, are susceptible to undesirable settling due to gravity. It is desirable to configure the cartridge and the assay process to maintain proper target component distribution within the sample.
Most prior assay cartridges are configured to perform multiple analyses on a single sample. It is also desirable to simultaneously perform one or more assays on multiple samples. Simultaneous processing of multiple samples requires consideration of certain time constraints when processing the assay. For example, when the concentration of the target component is determined by fluorescence emissions, the signal detected by the imaging instrument frequently varies with the period of time that the sample is contacted with the reagent. Therefore, it is important that all the samples be assayed using the same or similar time conditions.
It is also frequently desirable and/or necessary to dilute the biological sample in order to accurately detect the amount of target component present. For example, it may be necessary to dilute concentrated biological samples so that a fluorescent signal from the sample and reagent mixture falls within an easily detectable or linear range. However, the degree of dilution needed varies depending on the initial concentration of the component within the sample. It is, therefore, important to be able to accurately dilute the samples being analyzed.
To achieve sample dilution, prior art cartridges frequently have utilized complex designs which are costly and difficult to manufacture. The complexity found in the cartridges is in part due to the metering of the sample within the cartridge. If the sample metering is performed using small hand-held pipets, then the configuration of the cartridge may be simplified. Thus, it is desirable to eliminate the metering aspects of the cartridge so as to simplify the cartridge design. This also provides for variable dilution ratios.
When the cartridge comprises a self-contained dilution apparatus, various configurations of capillaries, conduits, chambers, reservoirs, application wells and stop junctions are used to move the sample, reagent and/or diluent within the cartridge. Prior art cartridges use capillary, gravitational and/or centrifugal forces to move the fluids within the cartridge. It has been disclosed to use capillary backpressure to create a "stop junction" which stops the flow of the fluids under certain conditions, while allowing flow under other conditions. Such stop junctions or stop flow capillaries act as valves without moving parts. The stop junctions are opened or "broken" by changing the pressure, force or acceleration applied to the fluid in the capillary forming the stop junction.
Prior art cartridges using a capillary to form a stop junction did not contemplate using low centrifugal accelerations to move the fluid past the stop junction. Prior art cartridges use high rotational speed or changes in fluid level in a reservoir to overcome the stop junction. In certain cartridge designs, variable fluid levels may be unavailable or undesirable. Similarly, high rotational speeds which may be desirable for separating plasma or similar components, can be detrimental to certain components within a sample. Thus, it is desirable to configure an assay or dilution cartridge which would move the sample, diluent and/or reagent by means of low centrifugal forces.
Various assay cartridges having dilution fluidics and various assay cartridges configured for centrifugal acceleration have been known for a number of years, and by way of example, several forms of such devices can be found in U.S. Pat. Nos. 4,728,500; 4,756,884; 4,946,795; 5,061,381; 5,122,284; 5,173,193; 5,186,844; 5,230,866 and 5,300,779. One system which incorporates the optics capable of using an assay cartridge is disclosed in co-pending U.S. patent application Ser. No. 08/236,342 entitled "Apparatus and Method for Volumetric Capillary Cytometry" invented by Thomas M. Baer, Louis J. Dietz, Robert S. Dubrow, Paul G. Hayter, Michael Hodges, Bala S. Manian and Robert J. Shartle, owned by the same assignee as this application and incorporated herein by reference. Similarly, a method and apparatus for gathering and analyzing data available from an assay cartridge is described in co-pending U.S. patent application Ser. No. 08/236,645 entitled "Method and Apparatus for Cell Counting and Cell Classification" invented by Ning L. Sitzo and Louis J. Dietz, also owned by the same assignee as this application and also incorporated herein by reference.
Hence, those concerned with the development and use of assay cartridges for the movement and dilution of fluid samples have long recognized the need for improved fluidic circuits. With the introduction of imaging instruments which use a diluted whole blood sample in a fixed volume, such as those described in the applications incorporated above, a need is now recocognized for systems which move the sample without subjecting the sample and the cartridge to high centrifugal accelerations. The present invention solves each of these needs.